Two individual biological replicates, each containing material of five mature plants of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, respectively, were used for the microarray analysis. Plants were germinated on soil and kept under continuous light until the beginning of bolting and then transferred to the dark for 8 h. Dark-incubated mature plants were reilluminated for 30 min and subsequently harvested for RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by using an RNeasy plant mini kit (Qiagen, Hilden, Germany) and quantified spectrophotometrically at 260 nm.
Label
Biotin
Label protocol
Only those samples with a 260/280 nm ratio between 1.8-2.1 and a 28S/18S ratio within 1.5-2 were further processed. Total RNA samples (5 mg) were reverse-transcribed into doublestranded cDNA with a One-Cycle cDNA synthesis kit (P/N 900431; Affymetrix, Santa Clara, CA). The doublestranded cDNA was purified using a Sample Cleanup Module (P/N 900371; Affymetrix). The purified doublestranded cDNAs were in vitro-transcribed in the presence of biotin-labeled nucleotides using a IVT labeling kit (P/N 900449; Affymetrix). The biotinylated c RNA was purified by using a Sample Cleanup Module (P/N 900371; Affymetrix), and its quality and quantity were determined with a NanoDrop ND 1000 and Bioanalyzer 2100.
Hybridization protocol
Biotin-labeled cRNA samples (15 mg) were fragmented randomly to 35-200 bp at 94°C in fragmentation buffer (P/N 900371; Affymetrix) and were mixed in 300 ml of hybridization buffer containing a hybridization control cRNA and control oligo B2 control (P/N 900454; Affymetrix), 0.1 mg/ml herring sperm DNA, and 0.5 mg/ml acetylated BSA in 2-(4-morpholino)-ethane sulfonic acid (Mes) buffer, pH 6.7, before hybridization to GeneChip Arabidopsis ATH1 genome arrays for 16 h at 45°C. Arrays were then washed using an Affymetrix Fluidics Station 450 EukGE-WS2v4_450 protocol. An Affymetrix GeneChip Scanner 3000 was used to measure the fluorescence intensity emitted by the labeled target.
Scan protocol
An Affymetrix GeneChip Scanner 3000 was used to measure the fluorescence intensity emitted by the labeled target.
Description
Two individual biologica replicates, each containing material of five mature plants of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu.
Data processing
Raw data processing was performed by using the Affymetrix GCOS 1.2 software. After hybridization and scanning, probe cell intensities were calculated and summarized for the respective probe sets by means of the MAS5 algorithm (1). To compare the expression values of the genes from chip to chip, global scaling was performed, which resulted in the normalization of the trimmed mean of each chip to a target intensity (TGT value) of 500 as detailed by Affymetrix. Quality control measures were considered before performing the statistical analysis. These included adequate scaling factors (between 1 and 3 for all samples), and appropriate numbers of present calls were calculated by application of a signedrank call algorithm (2). The efficiency of the labeling reaction and the hybridization performance were controlled with the following parameters: Present calls and optimal 3'/5' hybridization ratios (»1) for the housekeeping genes (GAPDH and ACO7), for the poly(A) spike in controls and the prokaryotic control (BIOB, BIOC, CREX, BIODN). After normalization, "baseline" samples were compared (i.e., wild type after a dark/light shift versus either flu or ex1/flu or ex2/flu or ex1/ex2/flu) and for the comparative analysis of changes in gene expression between wild type and others, only the genes that met the following criteria were considered: (i) The genes should show a reliable level of RNA giving a detection call of P (present in the Affymetrix nomenclature), and (ii) the signals should be changed by at least 2-fold or greater ("difference" call) relative to the baseline sample. Based on these criteria, the remaining genes were selected for analysis using Genespring 7.2 package. The microarray analyses were performed in duplicate using independent samples for wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu plants.
